Bioconjugate Chem. 1995, 6, 255-263
255
“Cleavable Trifunctional”Approach to Receptor Affinity Labeling: Chemical Regeneration of Binding to AI-Adenosine Receptors Kenneth A. Jacobson,* Bilha Fischer, a n d Xiao-duo Ji Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 . Received September 15, 1994@
A general approach for reversible affinity labeling of receptors has been developed. The objective is to carry out a series of chemical modifications resulting in a covalently-modified, yet functionallyregenerated, receptor protein that also may contain a reporter group. The ligand recognition site of AI-adenosine receptors in bovine brain membranes was probed to demonstrate the feasibility of this approach. Use of disulfide or ester linkages, intended for cleavage by exposure of the labeled receptor to either reducing reagents or hydroxylamine, respectively, was considered. Binding of the antagonist radioligand r3H1CPXwas preserved following incubation of the native receptor with 3 M hydroxylamine, while binding was inhibited by the reducing reagent dithiothreitol (DTT) with a n IC50 of 0.29 M. Hydroxylamine displaced specific agonist ([3HlPIA)binding in a noncovalent manner. Specific affinity labels containing reactive isothiocyanate groups were synthesized from XCC (8-[4-[(carboxymethyl)oxylphenyll-1,3-dipropylxanthine)and shown to bind irreversibly to Al-receptors. The ligands were structurally similar to previously reported xanthine inhibitors (e.g.,DITC-XAC: (1989) J. Med. Chem. 32, 1043) except that either a disulfide linkage or a n ester linkage was incorporated in the chain between the pharmacophore and the isothiocyanate-substituted ring. These groups were intended for chemical cleavage by thiols or hydroxylamine, respectively. Radioligand binding to AI-receptors was inhibited by these reactive xanthine5 in a manner that was not reversed by repeated washing. Hydroxylamine or DTT restored a significant fraction of the binding of [3HlCPX in AI-receptors inhibited by the appropriate cleavable xanthine isothiocyanate derivative.
INTRODUCTION
M i n i t y labeling is a widely used technique for the characterization of receptor proteins (I). It is particularly useful for membrane-bound proteins which are not readily characterized by other physical methods, due to the difficulty of isolation in quantities required. By the affinity labeling technique a high affinity ligand that contains a chemically reactive group, such as a bromoacetyl, methylfumaryl, or isothiocyanate (21, or a photochemically reactive group, such as azide (3), is synthesized. The electrophilic group of a chemicallyreactive ligand may spontaneously combine with a nucleophilic group that may be present on the binding site of the receptor ( 4 ) or on a neighboring molecule in the membrane (5). We have utilized a variety of cross-linking reagents to covalently anchor selective, purine ligands (amine-functionalized congeners) to subtypes of adenosine receptors (2,6, 7). Generally, receptor affinity labels have been designed without a detailed knowledge of the environment of the ligand binding site, and the identification of reactive ligands that bind covalently to the receptor protein has been empirical. A radioisotope may be incorporated into the affinity label (6,201 for purposes of receptor detection (e.g., on electrophoretic gels) and imaging. A deficiency of typical protein labeling is that the modified receptor, although intact in its primary structure, is functionally inactive, both in ligand binding and in activation of second messengers. It would be desirable to have a general means of regenerating the functional binding site following affinity labeling, either in its native form or in a covalently-labeled state. Thus, several
* To whom correspondence should be addressed at Bldg. 8A, Rm. B1A-17, NIDDK, National Institutes of Health, Bethesda, MD 20892. Tel.: (301)496-9024. Fax: (301)402-0008. Abstract published in Advance ACS Abstracts, March 15, @
1995.
groups have introduced reversible affinity labeling schemes (8, 9, 23). Patchornik et al. (8)designed a scheme for the photochemical deblocking of an affinity-labeled biopolymer to regenerate its native form. Denny and Blobel (9) reported a n lz5I-labeledheterobifunctional protein crosslinking reagent (N-[4-[(p-azido-m-iodophenyl)azolbenzoyll-3-(aminopropyl)-N‘-oxysulfosuccinimide ester) that may be cleaved by the action of sodium dithionite on a n azo linkage joining the two reactive moieties. The biopolymer is not regenerated in its native form; instead a radioiodine label remains with one component after cleavage. Similarly, disulfide groups are used in a variety of protein and nucleic acid cross-linkers (IO), such a s biotinylated probes, for subsequent cleavage under reducing conditions. Adenosine is a neuromodulator that allows organs such as the heart, brain, and kidneys adapt to stress and maintain homeostasis, by acting a t specific cell surface G-protein coupled receptors (reviewed in ref 21). As part of a detailed structural investigation of AI- and Azaadenosine receptor subtypes, we introduced a general trifunctional approach to affinity labeling (11). By this approach a reporter group (e.g.,radioactive or spectroscopic) is tethered to a receptor ligand, which delivers it to the binding site selectively, and then incorporated covalently onto the receptor protein via a chemically reactive group (an isothiocyanate) present on the same molecule. A key intermediate is a trifunctional crosslinker consisting of a 3,5-diisothiocyanatobenzenederivative (equivalent to structure 2 in Figure 1). One of the isothiocyanate groups reacts with a n amine-functionalized ligand, and the other reacts with the receptor protein. In this study we have expanded this approach conceptually to include chemically-cleavable spacer groups. An amine-functionalized ligand, 1, may be designed t o contain a cleavable A-B linkage (Figure 1). Upon coupling to a trifunctional cross-linker, 2, a conjugate 3
1 043-1802/95/2906-0255$09.00/0 0 1995 American Chemical Society
Jacobson et al.
256 Bioconjugafe Chem., Vol. 6,No. 3, 1995
1) Reactswith
I
functionalized drug congener
2) Reacts with a nucleophile on the receptor
3
R
4a
R
4b
Figure 1. General scheme for reversible affinity labeling that leaves a reporter group on a receptor protein. Structure 1 is a n amine-functionalized congener that contains a cleavable (A-B) linkage. A conjugate, 3, is formed with a trifuctional cross-linking reagent (structure 2), which may be used to affinity label a receptor (as in 4a). The A-B linkage is then cleaved (structure 4b), which allows the free ligand containing the pharmacophore to diffuse away from the binding site.
min the solvent was evaporated, and 1M NaHzP04 was added (10 mL, pH 4.2). The aqueous solution was extracted with ether to remove the di-t-Boc-cystamine (mp 106-107 "C, 0.135 g, 17.5%). The aqueous solution was basified to pH 9 by 1 M NaOH and extracted with EtOAc (5 mL x 6). The combined organic phases were dried over MgS04 and evaporated to yield the product (0.24 g, 43%). The product was isolated as the hydrochloride salt (mp. 109-110 "C). 'H NMR (DzO) 6: 3.40 (q, 4H, CHZN, J = 6.6 Hz), 3.00 (q,2H, CHzS, J = 7 Hz), 2.87 (t, 2H, CHZS, J = 6.3 Hz), 1.45 (s,9H, t-BuO). MS (CUNH3) free amine: 253 (MH+) 253 (MH+ - t-Boc). EXPERIMENTAL PROCEDURES Anal. Calcd. for CgHzlClNzOzSz: C, 37.42; H, 7.33; N, 9.70. Found: C, 37.46; H, 7.34; N, 9.61. Synthesis. IH NMR spectra were recorded using a 8-~4-[[[~[1-[2-[(tert-Butyloxycarbonyl)amimlethylldithwlVarian XL-300 FT-NMR spectrometer, and all values are ethyl]amino]carbonyl]methylloxylphenyl]-l,3-dipropylreported in parts per million (ppm, 6) downfield from xanthine, 10. Mono-t-Boc-cystamine ( 8 , 80 mg, 0.32 tetramethylsilane (TMS). Chemical ionization MS using mmol), EDAC (0.165 g, 0.85 mmol), and l-hydroxybenionized NH3 gas were recorded using a Finnigan 1015D zotriazole (0.1 g, 0.75 mmol) were added to XCC (9,0.125 mass spectrometer modified with EXTREL electronics. g, 0.32 mmol) in dry DMF (10 mL). The reaction mixture Fast atom bombardment MS was carried out on a JEOL was sonicated a t room temperature for 30 min. EtOAc JMS-SXlO2 mass spectrometer. Thin-layer chromatog(10 mL) was added, and the turbid solution was extracted raphy (TLC) analyses were carried out using EM Kiewith water and then with 2 M NaZC03 (pH 11). The selgel 60 F254, DC-Alufolien 200 xb5 plates and were combined aqueous solutions were extracted with EtOAc visualized under ultraviolet light. Elemental analyses (10 mL x 21, dried, and evaporated under high vacuum. were performed by Atlantic Microlabs, Inc., Atlanta, GA. The solid obtained was washed with a small amount of XCC,l m-DITC-XAC, and m-DITC-ADAC were syntheether and dried under high vacuum to yield a light sized as previously reported (2,16). Cystamine dihydroyellowish solid (0.147 g, 75%), mp 184 "C. IH NMR chloride was obtained from Sigma (St. Louis, MO). (CDC13) 6: 8.23 (d, 2H, ArH, J = 8.8 Hz), 7.05 (d, 2H, N-tert-Butyloxycarbonyl)cystamine,8. Di-tert-butyldiArH, J = 8.8 Hz), 4.95 (br s, l H , NH), 4.60 (s, 2H, CHzO), carbonate (0.48 g, 2.2 mmol) and triethylamine (0.91 mL, 4.19, 4.11 (t, 2H, CHzN), 3.72, 3.43 (2H, CHzNH), 2.90, 3 equiv) were added to a methanolic solution (25 mL) of 2.82 (t, 2H, CHzS), 1.88, 1.77 (9, 2H, CHzCHz), 1.45 (s, cystamine bis hydrochloride (0.5 g, 2.2 mmol). After 20 9H, t-Boc), 1.02, 0.99 (t, 3H, CHzCH3). MS (FAB): 621 Abbreviations: ADAC, N6-[4-[[[[4-[[[(2-aminoethyl)aminol- (M+). Anal. Calcd. for CzsHroNsO6Sz: c, 54.18, H, 6.49; N, 13.54. Found: C, 54.28; H, 6.54; N, 13.47. carbonyllmethyllanilinolcarbonyllmethyllphenylladenosine;Boc, tert-butyloxycarbonyl; Cbz, benzyloxycarbonyl; CPX, 8-cyclo8-[4-[[[[[1-[(2-Aminoethy1)dithio/ethy11amino]carbony11pentyl-1,3-dipropylxanthine; DITC, phenylene diisothiocyanate; methyl]oxy]phenyl]- 1,3-dipropylxanthine Trijluoroacetate, DTT, dithiothreitol; EDAC, N - e t h y l 4 '-(3-diaminopropyl)car11. Compound 10 (0.047 g, 0.756 mmol) was dissolved bodiimide hydrochloride; EtOAc, ethyl acetate; IBMX, 3-isobuin trifluoroacetic acid (1.5 mL), and the solution was tyl-1-methylxanthine; PIA, W-phenylisopropyladenosine; TFA, stirred a t room temperature under nitrogen. After 10 trifluoroacetic acid; Tris, tris(hydroxymethy1)aminomethane; min, the flow rate of nitrogen was increased, and triXAC, 8-~4-~~~~~2-aminoethyl~aminolcarbonyllmethyllo~lphenyllfluoroacetic acid was evaporated. Ether (5 mL) was 1,3-dipropylxanthine; XCC, 8-[4-[[(carboxymethyl)oxylphenylladded to the glassy residue to form a white solid, which 1,3-dipropylxanthine. is obtained. In 3 the cleavage site is located between the trifunctional phenyl ring and the pharmacophore. After receptor binding and cleavage of A-B, a portion of the label remains covalently bound to the receptor protein (structure 4b). The attached portion contains the reporter group (R) but not the pharmacophore moiety. The pharmacophore may then freely dissociate from the binding site. Thus, the receptor protein remains chemically labeled, and in principle, the receptor binding site is, a t least in part, unoccupied and again able to bind radioligands.
Bioconjugate Chem., Vol. 6,No. 3, 1995 257
Cleavable Affinity Labeling of A1-Receptors
8-~4-~~~~~1-[~~2-~(Benzy1oxycarbony1~amino1ethy11oxy1 was dried under high vacuum for 48 h (0.044 g, 92%), carbonyllethyllaminolcarbonyllmethylloxylphenyll-l,3mp > 230 "C. 'H NMR (CD30D)6: 8.02 (d, 2H, ArH, J dipropylanthine, 17. Compound 16 (0.15 g, 0.39 mmol), = 8.7 Hz), 7.13 (d, 2H, ArH, J = 8.7 Hz), 4.83 (8, 2H, EDAC (0.4 g, 1.8 mmol), and 1-hydroxybenzotriazole OCHzCO), 4.13, 3.97 (t, 2H, CHzN, J = 7 Hz), 3.63 (t, (0.05 g, 0.37 mmol) were added to XCC (9, 0.12 g, 0.31 2H, CHZNHZ), 3.50 (q,2H, CHzNH, J = 7 Hz), 2.98, 2.91 mmol) in dry DMF (20 mL). The reaction mixture was (t, 2H, CHzS), 1.75, 1.64 (9, 2H, CHzCHz), 0.99, 0.95 (t, sonicated at room temperature for 30 min. Water was 3H, CHzCH3). Anal. Calcd. for C Z ~ H ~ ~ N ~(hydroO&F~ added, and a precipitate formed. The solid obtained was trifluoroacetate of 11>1/2Hz0: C, 46.65; H, 5.32; N, 13.06. washed with water and dried under high vacuum (0.111 Found: C, 46.67; H, 5.26; N, 12.77. g, 56%), mp 204-205 "C (heated slowly). The product 8-[4-~[[~~1-~~2-[~K3-Isothiocyanatophenyl~aminolthio17 was homogeneous by TLC. carbonyllami~lethylldithiolethyllamino/ Anal. Calcd. for C ~ Z H ~ ~ N ~ O C, ~ 59.71, - ~ / ~H,H6.11; Z~: oxy]phenyl]-l,3-dipropylxanthine,12. A dry DMF soluN, 13.06. Found: C, 59.72; H, 5.98; N, 13.15. tion (5 mL) of product 11 (0.032 g, 0.053 mmol), 8-[4-[[[[[1-[[(2-Aminoethyl)oxy]carbonyl]ethyl]amino]triethylamine (0.1 mL, 2 equiv), and m-phenylene dicarbonyllmethylloxylpheny11-1,3-dipropylxanthine Hydro isothiocyanate (ref 6,30 mg, 3 equiv) was stirred a t room bromide, 18. Compound 17 (26.6 mg, 42 xb5mol) was temperature for 0.5 h. The solvent was removed under dissolved with vortexing in 30% HBrIacetic acid (1mL), high vacuum (bath temperature 36 "C) to leave a and the solution was stirred a t room temperature under semisolid residue, which was purified on a micro silica nitrogen for 30 min. The flow rate of nitrogen was column (EtOAc 1%Et3N),followed by treatment of the increased leaving a n oil, and the product was precipitated product with dry ether and drying under high vacuum as a microcrystalline solid from methanovether. The (30 mg, 84%), dec 185 "C. The product was homogeneous supernatant was removed with a Pasteur pipette, and by TLC (Rf = 0.82, silica, using ch1oroform:methanol: the remaining white solid was washed (ether, 3x1 and acetic acid 85:10:5, by vol) and reacted with ethylenedidried under high vacuum (25 mg, 100%),mp 273 "C dec. amine to form cleanly a new compound of Rf 0.30 in the The product was homogeneous by TLC (Rf= 0.78, silica, 8.93 (s, lH, ArH-21, same system. lH NMR (CDCld 6: using ch1oroform:methanol:acetic acid 10:10:1, by vol). 8.16 (d, 2H, ArH, J = 8.7 Hz), 7.34 (m, l H , ArH), 7.04 MS: 501 (M l), 483,329. 'H NMR (DMSO-de)6: 8.29 (d, 2H, ArH, J = 8.7 Hz), 6.96 ('t', 2H, ArH, J = 5.2 Hz), (t, H, CONH), 8.08 (d, 2H, J = 8.8 Hz, 8-ArH, ortho), 4.60 (s,2H, CHzO), 4.17 (t, 2H, CHzN), 3.73 (q,2H, CHz7.83 (br s, 2H, NHz), 7.09 (d, 2H, J = 8.8 Hz, 8-ArH, NH), 3.14, 3.12 (q, 2H, CHzN), 3.02, 2.87 (t, 2H, CHzS), meta), 4.57 (s, 2H, CHZO), 4.20 (t, 2H, CHz), 4.02, 3.87 1.86, 1.71 (9, 2H, CHzCHz), 1.01, 0.96 (t, 3H, CHzCH3). (each, t, 2H, J = 7 Hz, Pr, CHZ), 3.40,3.09 (each: m, 2H, Anal. Calcd. for C31H36N80&: C, 52.23; H, 5.09; N, CH2), 2.57 (t, 2H, J = 6.9 Hz, CHd, 1.74, 1.58 (each: q, 15.72. Found: C, 51.80; H, 5.26; N, 15.13. 2H, Pr, CHz), 0.89 (m, 2 x 3H, Pr, CH3) ppm. 2-[(Benzyloxycarbonyl)amino]ethanol, 13. 2-Aminoeth8-[4-[[[[[1-[[[2-[[[[(3Isothiocyanatop henyl)amino]thioIanol(O.91 g, 15 mmol) was dissolved in ethyl acetate (30 carbonyl]amino]ethyl]oxylcarbonyl/ethyllaminolcarbonyllmL), and sodium carbonate (3 g) was added. The mixture methyl]oxy]phenyl]-1,3-dipropylxanthine, 19. Compound was treated with a solution of benzyl chloroformate (3.3 19 was synthesized in 91% yield from compound 18 and g, 19 mmol) dissolved in 30 mL of ethyl acetate, added m-phenylene diisothiocyanate by the general method in aliquots with stirring. The mixture was filtered, and given for compound 12. The product was recrystallized the filtrate was treated with water and ethyl acetate. from DMF/ether, was homogeneous by TLC (Rf= 0.84, After separation of the layers, the organic layer was silica, using ch1oroform:methanol:aceticacid 85:10:5, by extracted with pH 7 phosphate buffer and then with 0.1 vol), and reacted with ethylenediamine to form cleanly M HC1. The organic layer was evaporated to dryness a new compound of lower Rf. lH NMR (DMs0-d~) 6: 9.80 leaving a residue which was triturated with petroleum (s, lH, ArNH), 8.23 (m, 2H, carbonyl-NH), 8.08 (d, 2H, ether. The resulting solid was collected and recrystal8-ArH, ortho), ArNCS: 7.60 (lH, HG),7.34 (2H, H z , ~ 7.15 ), lized fron ethyl acetate/petroleum ether to provide the (lH, H3), 7.06 (d, 2H, 8-ArH, meta), 4.55 (s, 2H, CHzO), product as white crystals (mp 58-59 "C, 36% yield). 4.19 (m, 2H, CHz), 4.01 (t, 2H, Pr, CHz), 3.87 (t, 2H, Pr, MS: 214, 196 (M 1). The NMR spectrum was consisCHz), 3.73, 3.62, 3.15 (each: 2H, CHz), 3.02, 2.87 (each: tent with the assigned structure. t, 2H, CH2), 1.74, 1.58 (each: q, 2H, Pr, CHd, 0.88 (m, 2 (tert-Butyloxycarbonyl)-P-alanine 2-[(Benzyloxycarbonx 3H, Pr, CH3) ppm. y1)aminolethyl Ester, 15. A solution of (tert-butyloxycar8-[4-[[[[[1-[[[2-[[[[3-Isothiocyanato-5-[[2-[[3-(4hydroxybonyl)-P-alanine (14,0.98 g, 5.2 mmol) and compound 13 p henyl)propionyl]amino]ethyl]aminolcarbonyllphenyll(0.81 g, 4.15 mmol) in DMF (40 mL) was treated with aminolthiocarbonyllamino/ethylloxylcarbonyllethyllami~lEDAC (1.3 g, 6.8 mmol) and DMAF' (0.8 g, 6.6 mmol) with carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine, 20. stirring. After 2 h, half-saturated sodium chloride was Compound 18 (8 mg, 14 pmol) and 1-[[3-(4-hydroxyadded. The product was extracted into ethyl acetate, phenyl)propionyl]amino]-3,5-diisothiocyanatobenzene (ref which was washed with 0.1 M HC1 and then 0.1 M Naz11, 19 mg, 45 pmol) were dissolved in 1mL of DMF, and COS,dried (NaZSOd), and evaporated, leaving an oil (0.96 diisopropylethylamine (5 pL) was added with stirring. g, 63% yield). MS: 384,367 (M 11,328,311,267. The After 1h, all of the amine (Rr= 0.08) had been consumed, NMR spectrum was consistent with the assigned strucas judged by TLC (silica, using ch1oroform:methanol: ture. acetic acid 85:10:5, by vol). Ether was added, causing a n oil to separate. The supernatant was removed, and P-Alanine 2-[(Benzyloxycarbonyl)amino]ethylEster Trithe residue was crystallized from MeOWether. The solid fluoroacetate, 16. Compound 15 (0.51 g, 1.4 mmol) was was washed with ether and dried under vacuum to dissolved in a minimum volume of trifluoroacetic acid, provide 8.6 mg of product (66% yield), which was homoand the solution was stirred a t room temperature under geneous by TLC (Rf= 0.51, same system as above). lH nitrogen. After 30 min, the trifluoroacetic acid was NMR (DMSO-dd 6: 10.0 (s, l H , ArNH), 8.7 (br s, l H , evaporated, and the glassy residue was dried under high OH), 8.59, 8.25 (each: t, l H , NH), 8.08 (d, 2H, J = 8.8 vacuum (0.33 g, 63%yield). MS: 267 (M + 11, 240, 212. Hz, 8-ArH, ortho), 7.94, 7.81 (each: m, lH, NH), 7.76, The NMR spectrum was consistent with the assigned 7.59 (each s, lH, ArNCS), 7.07 (d, 2H, J = 8.8 Hz, structure.
+
+
+
+
Jacobson et al.
258 Bioconjugafe Chem., Vol. 6,No. 3, 1995
8-ArH, meta), 6.96,6.64 (each, d, 2H, J = 8.3 Hz, ArOH), 4.55 (s,2H, CHZO), 4.19 (t, 2H, J = 5.3 Hz, CHz), 4.01 (t, 2H, J = 6.9 Hz, Pr, CHz), 3.86 (t, 2H, J = 7.3 Hz, Pr, CHz), 3.74,3.40,3.25,3.20 (each m, 2H, CHd, 2.68,2.55, 2.29 (each: t, 2H, CH2),1.74, 1.58 (each: q, 2H, Pr, CHd, 0.89 (m, 2 x 3H, Pr, CH3) ppm. Binding Assays. Bovine cerebral cortical membranes were prepared as described previously (12),from frozen brains obtained from Pel-Freeze Biologicals Co. (Rogers, Arkansas). Membranes were treated with adenosine deaminase (0.5 U/mL) for 20 min a t 37 "C prior to radioligand binding studies or incorporation studies. Inhibition of binding of 1nM [3Hl-l\rs-phenylisopropyladenosine or 0.2 nM [3H]-8-cyclopentyl-l,3-dipropylxanthine (Dupont NEN, Boston, MA) to AI-adenosine receptors in bovine cerebral cortex membranes was assayed as described (13,14). Membranes (40 pg, 150 pL) were incubated for 1 h a t 25 "C in a total volume of 1 mL, containing 100 pL of radioligand of the indicated concentration and 25 pL of the competing ligand (dissolved as a stock solution in DMSO). Samples of the drugs were dissolved freshly from solid and stored a t -80 "C. The DMSO solutions were diluted to a concentration of less than 0.1 mM prior to adding to aqueous medium. Bound and free radioligand were separated by addition of 4 mL of a solution containing 50 mM Tris hydrochloride, a t pH 7.4 a t 5 "C, followed by vacuum filtration on glass filters with additional washes totaling 12 mL of buffer. Nonspecific binding was determined with 10 pM 2-chloroadenosine or 100 pM W-cyclopentyladenosine. Protein was determined using the BCA protein assay reagents (Pierce Chemical Co., Rockford, IL). All competition binding data was analyzed by nonlinear regression using the InPlot computer program (GraphPAD, San Diego, CAI, and IC50 values were converted to Ki values using the Cheng-Prusoff equation (25). The Kd values, used in these calculations for L3H1PIA and r3H1CPX binding to bovine brain Al-receptors were 130 f 4 pM and 74 f 3 pM, respectively (13). To assay for irreversible inhibition, a n incubation at 37 "C for 1h in 50 mM Tris pH 7.4 with an isothiocyanate derivative was carried out and optionally followed by a second incubation in 50 mM Tris pH 7.4 with DTT or hydroxylamine to test for chemical reversibility. The incubation with DTT was always carried out a t room temperature, and the incubation with hydroxylamine was either a t rt or 37 "C (latter preferrable). Washing cycles for inhibition experiments between incubations involved three cycles of centrifugation and resuspending the membrane pellet by stirring or homogenization using the Polytron. At the final step, prior to radioligand binding, the membranes were homogenized using a glass tissue grinder. RESULTS
In order to demonstrate the conceptual approach of Figure 1, it was necessary to identify linkages (A-B in amine congener 1)that may be cleaved chemically using reagents that are not detrimental to adenosine receptors and that would be compatible with an aqueous medium. Two likely possibilities were the reduction of disulfide bonds by thiols and the aminolysis of ester bonds by hydroxylamine. Receptors and other proteins, even those containing structurally important disulfide bridges, may be exposed to either thiol reagents or hydroxylamine below determined concentration limits and remain active (7). The stability of adenosine receptors to potential cleavage conditions a t room temperature was examined. The effects of chemical reagents on radioligand binding were
Table 1. Stability of Bovine ApAdenosine Receptors to Reagents for Use in Cleavage Reaction: (A) at Fixed Concentration with Intervening Wash" and (B) Concentration Dependence of Inactivation of Receptor Binding by Reagents for Potential Regeneration, with and without Intervening Washb specific binding remaining (% of control) L3H]CPX L3H1PIA
(A) reagent none D'I"l' (5 mM) DTT (50 mM) HzNOH (250 mM)
100 97 f 19 98 i 2 70 i 8
100 98 f 3 98 f 2 96 f 5
ICs0 (mM) or % inhibition (at concn indicated) L3HlCPX L3H1PIA (B) reagent wash:
+
+
DTT HzNOH
290f37 0% (2 M)
*
158&42 0% (3 M)
3 0 8 f 17 15%(2 M)
85*8 385 f 144
a Data are means SE of three to four experiments. Dithiothreitol or hydroxylamine was added during a 1 h preincubation with membranes at room temperature prior to radioligand binding. The concentrations of radioligand used were 0.2 and 1.0 nM for L3H]CPX and PHIPIA, respectively. Data are means f s.e.m. of four to five experiments. Dithiothreitol or hydroxylamine was added either (1)during a 1 h preincubation with membranes at room temperature and removed by washing or (2) during the radioligand binding. The concentrations of radioligand used were 0.2 and 1.0 nM for L3H]CPX and I3H1PIA, respectively.
*
measured in two different ways (Table 1): (1) as a preincubation with membranes followed by a washing step prior to radioligand binding and (2) addition of the reagent to the binding assay medium in the presence of the radioligand. Any difference between the two results might represent reversible competition by the highly concentrated chemical reagent for the radioligand binding site. The stability of A1 receptors to potential chemical cleavage conditions has not been reported. By analogy with a closely related protein sequence, our previous study of rabbit Az, receptors demonstrated moderate stability of the receptor to 5 mM dithiothreitol (DTT) or 250 mM hydroxylamine (7). However, Aza receptors potentially have more disulfide bridges (four have been proposed (24))than do A1 receptors; thus, it was necessary to test the stability ofAl receptors to these reagents. Bovine brain membranes containing A1 adenosine receptors were exposed for 60 min to either a thiol or hydroxylamine and then washed and subjected to radioligand binding. It was observed that a t a 5 mM concentration of DTT, the binding of L3H1CPX,a n AI-selective antagonist, and L3H]PIA, a n AI-selective agonist, was maintained near control levels (Table 1A). The stability of the receptor to hydroxylamine was even more striking, with a concentration of 250 mM tolerated. The limits of stability a t yet higher concentrations were probed (Table 1B). At room temperature, concentrations of hydroxylamine up to 3 M (maximum allowed by solubility) were well tolerated by the receptor, especially when [3HlCPXwas used in the subsequent binding assay. Without an intervening wash, the IC50 values for DTT inhibition of radioligand binding were 158 mM for [3H]CPX and 85 mM for [3H]PIA binding. When DTT was removed prior to the binding assay, ICs0values increased to 290 mM for [3HlCPXand 308 mM for I3H1PIAbinding. The latter values more closely reflect the effects of DTT on the receptor protein, which likely contains disulfide linkages. When the hydroxylamine remained in the medium concurrently with the radioligand, agonist binding alone was adversely affected. The ICs0 value for hydroxylamine inhibition of f3H1PIA binding was 385
Cleavable Affinity Labeling of A,-Receptors
Bioconjugafe Chem., Vol. 6,No. 3, 1995 259
Chart 1
.NCS
H
HO OH
Table 2. Inhibition of Bovine AI-Adenosine Receptors by Purine Isothiocyanate Derivatives (without Cleavable Chains) and the Stability of this Inhibition to Reagents for Later Use in Cleavage Reaction" inhibition (% of control) n-DITC-XAC, 5 concn reagent (mM) L3H1CPX L3H1PIA none 80 f 4 (7) 55 f 7 (3) DTT 5 82f4(8) 55f4(4) 50 78,81 DTT nd HzNOH 250 78 f 3 (9) 61 f 8 (9)
n-DITC-ADAC, 6 L3H1CPX 48 f 8 (6) 50f4(8) 52,58 47 f 6 (6)
L3H1PIA 51 f 4 (4)
52i11(4) nd 50 f 12 (6)
a Data are means i s.e.m. of three to nine experiments (ngiven in parentheses). After incubation with the isothiocyante derivative (5 or 6) at 37 "C, membranes were washed three times, incubated overnight with IBMX (200 pM) in the presence of the indicated reagent, and again washed three times with IBMX and twice for DTT or hydroxylamine treatment. Radioligand binding was carried out with 0.2 nM [3H]CPX(14)or 1 nM [3H]PIA (13).nd: not determined.
mM. Curiously, with a n intervening wash, even a concentration of hydroxylamine of 2 M resulted in only 15% inhibition of [3H]PIA binding. This suggests that high concentrations of hydroxylamine interact noncovalently with a site on the receptor or on the radioligand that interferes with agonist binding alone. This complication notwithstanding, the use of hydroxylamine is acceptable in the scheme proposed in this study, because a washing step may be included and because an antagonist radioligand alone may suffice to demonstrate the feasibility of the cleavage scheme. The next step was to identify purine derivatives that may be employed in this scheme. The previous trifunctional study (21) utilized a series of functionalized xanthine derivatives, based on the A1 antagonist XAC (8-[4[[[[(2-aminoethyl)aminolcarbonyllmethylloxylphenyll1,3-dipropylxanthine), that acted a s irreversible AIinhibitors a t concentrations in the range of 10-7-10-6 M. We have also shown that several isothiocyanate derivatives of ADAC, a n agonist with selectivity and subnanomolar affinity for A1 adenosine receptors, acted as irreversible inhibitors of the receptor a t concentrations in the range of lo-* M. We reexamined the compounds m-DITC-XAC, 5, and m-DITC-ADAC, 6, as irreversible Al-inhibitors in bovine brain membranes (Chart 1, Table 2). It is to be noted that these ligands are analogous to structure 3 in Figure 1in which R = H, except that they are lacking the cleavable group A-B. A 1 h incubation with 100 nM m-DITC-XAC or with 10 nM m-DITCADAC resulted in inhibition of 80 or 48% of the L3H1CPX binding, respectively. This inhibition was not reversible upon repeated washing of the membranes.
It was necessary to demonstrate that the receptor inhibition was not reversible under the conditions intended to be employed for the cleavage step (i.e., exposure to DTT or hydroxylamine). If binding ability of the receptor were restored, as was found in a study of affinity labels for A2,-adenosine receptors (71, it would indicate that the site of reaction between the isothiocyanate group and the receptor protein would be sensitive to these chemical reagents. The inhibition was stable, as summarized in Table 2. Neither DTT (50 mM) nor hydroxylamine (250 mM), present during a second incubation after removal of the affinity label, reversed this inhibition. In the previous study of irreversible inhibitors of AIadenosine receptors ( 2 )based on XAC and ADAC, it was observed that the chain length separating the pharmacophore and the reactive electrophilic group (an isothiocyanate) could be varied somewhat without loss of the irreversible binding feature of the ligand. Thus, it was reasonable that a chain extension to include a cleavable linkage (A-B) would not preclude covalent binding to the Al-receptor. The antagonist series, rather than the agonist series, was developed in the subsequent compounds to avoid ambiguity, since agonist binding is subject to modulation by the state of coupling between the receptor and G-protein. Also, the binding of the antagonist i3H1CPXis less sensitive than L3H1PIAbinding to the presence of hydroxylamine. Thus, we designed several new amine congeners related to XAC, in which the terminal ethylenediamine moiety of the chain was extended by the CHzABCHz group placed in the middle. AB consisted of either SS (thiol cleavable), 11 (Figure 2)) or COO (hydroxylamine cleavable), 18 (Figure 3). These amine congeners corresponded to structure 1 in the general scheme (Figure 1). Each amine congener was to be coupled to a bifunctional o r trifunctional crosslinking reagent to form a potentially cleavable affinity label (structure 3). The amine congeners were synthesized from a xanthine carboxylic congener, 9 (XCC, 8-[4-[(carboxymethyl)oxylphenyl]-1,3-dipropylxanthine),which also served a s a n intermediate in the synthesis of XAC (16).The cysteamine (HS(CH2)zNHz)conjugate of XCC, a thiol derivative, was prepared previously and found to be a potent adenosine antagonist ( I 7). The Ki value of the cysteamine conjugate of XCC (17) was determined to be 16 nM vs [3H]PIAa t rat A1 receptors. That conjugate was also shown to be biologically active in a functional assay, in the inhibition of adenosine agonist-induced stimulation of adenylate cyclase via Az,-receptors, with a KB value was 76 nM (I7). Cystamine (compound 7 ) is a disulfide dimer of cysteamine. The conjugate of XCC and cystamine (compound 11, Figure 2), an amine congener, was
Jacobson et al.
260 Bioconjugate Chem., Vol. 6,No. 3, 1995
10
12 R = H
Figure 2. Synthesis of a thiol-cleavable xanthine amine congener, 11,containing a disulfide linkage. The final step consisted of reaction with m-phenylene isothiocyanate to form 12.Reagents: (a) di-tert-butyl dicarbonate; (b) EDAC/l-hydroxybenzotriazole, DMF; (c) TFA (d) m-phenylene diisothiocyanate (R = H) or derivative thereof.
15
13
VNY AN I
O
9
16
0
~ ~ ~ J + ~ Hf i N H c o ~ c H ~ ~
17
\
O
Y
0
-+!-o~NbcW./4Nn.
ti
OAN 18
I9 R=H 20 R L CONH(CH&NHCO(CHd&pOH
Figure 3. Synthesis of a hydroxylamine-cleavable xanthine amine congener, 18,containing an ester linkage. The final step consisted of reaction with m-phenylene isothiocyanate to form 19 or with 4-[2-[[[2-[[3,5-diisothiocyanatobenzoyllamino]ethyl]aminolcarbonyllethyl]-2-iodophenol to form 20. Reagents: (a) EDACDMAP, DMF; (b) TFA (c) EDACI1-hydroxybenzotriazole,DMF; (d) HBr/acetic acid; (e) m-phenylene diisothiocyanate (R = H) or derivative thereof.
isolated previously as a byproduct in the synthesis of the cysteamine derivative. In this study compound 11 was synthesized by condensing XCC,9,with Boc-cystamine, 8,followed by deprotection using TFA (Figure 2). The Ki value of the disulfide compound 11 was found to be 10 nM vs [3H]PIA at rat AI receptors, indicating that chain extension was not detrimental to binding affinity. The ester-containing amine congener (compound 18, Figure 3) was synthesized by condensing XCC with the
@-(Cbz-amino)ethylester of @-alanine, 16,followed by deprotection using HBr/acetic acid. The next step was to couple compounds 11 and 18 to diisothiocyanate derivatives and to show that the conjugates would irreversibly bind to bovine A1 receptors. The amine derivatives reacted with m-phenylene diisothiocyanate to form compounds 12 and 19, respectively. The Ki values for 12 and 19 in the displacement of L3HICPX in a “competitive” assay were found to be 2.1
Cleavable Affinity Labeling of A1-Receptors
Bioconjugafe Chem., Vol. 6,No. 3, 1995 261
Table 3. Irreversible Inhibition of Radioligand Binding by Compound 12 and Reversal of the Inhibition by DTT" concn of DTT (mM) 0 5 50
100
r3H1CPX
PHIPIA
% inhibn
% reversal
% inhibn
% reversal
63 f 5 57 f 5 48 f 4 38 f 4
6 15 25
69 f 5 52 f 1 44 f 7 40 f 3
17 25 29
a Data are means i s.e.m. of three to 11 experiments, expressed as percent of control binding. After incubation with the isothiocyante derivative 12 (500 nM) for 1 h at 37 "C, membranes were washed three times, incubated with DTT for 60 min at room temperature, washed twice, and then incubated with 0.2 nM PHICPX ( 1 4 )or 1 nM r3H1PIA (23).
Table 4. Relationship between Concentration of Compound 19 and the Ability of Hydroxylamine to Restore Binding of t3H1CPX concn of 19 (nm) 100 250 500
inhibition of [3H]CPX binding (% control) (-HzNOH) (+HzNOH) 54.4 63.3 i 1.2 76.0 i 3.0
Table 5. Relationship between Temperature and the Regeneration by Hydroxylamine of [3H]CPXBinding Following Inhibition by Compound 19
34.5 43.6 f 4.9 54.0 f 4.0
concn of 19 (mM) 250 500
a Data are means f s.e.m. of three experiments, unless noted. After incubation with the isothiocyanate derivative 19 at 37 "C for 1 h, membranes were washed three times, incubated with hydroxylamine (500 mM) at 37 "C for 1 h, washed three times, and then incubated with 0.2 nM r3H]CPX ( 1 4 ) . Regeneration following overnight incubation with hydroxylamine at room temperature resulted in regeneration comparable to 1h at 37 "C (data not shown).
I"
h
x
v
15-
reversal of inhibition (% control)
m
20 20 22
.-5
Data are means i s.e.m. of three experiments or a single experiment. After incubation with the isothiocyanate derivative at 37 "C for 1 h, membranes were washed three times, incubated with hydroxylamine (500 mM, 37 "C for 1 h), washed twice, and then incubated with 0.2 nM [3HlCPX ( 1 4 ) . When the hydroxylamine incubation was carried out at 25 "C for 1 h, the degree of reversal was 8% (100 nM 191,9% (250 nM 191,or 15% (500 nM 19). a
f 0.26 and 9.7 f 3.5 nM, respectively. Thus, receptor affinity has been preserved. In a n assay of irreversible binding, both 12 and 19 were shown to be effective inhibitors (Tables 3 and 4), comparable in effectiveness to m-DITC-XAC, 5. The ability of DTT or hydroxylamine, present during a second incubation, to reverse this inhibition by compound 12 or 19,respectively, was examined. Compound 12 a t a concentration of 500 nM caused a loss of 6369% of the radioligand binding (Table 3). This binding was partially restored (recovered 15-25% relative to control level) upon exposure to DTT. At 100 mM DTT the reversal of inhibition of the antagonist binding site was more effective than a t 50 mM. In Table 2 and in our previous study (2),3-isobutyl-lmethylxanthine (IBMX) was added to the washing medium as a precaution for the removal of noncovalently bound xanthine. In the present study it was not necessary to wash the membranes overnight with IBMX. A comparison of the reversal of inhibition by compound 12 (preincubation a t 500 nM) using DTT (10,50 or 100 mM) overnight a t room temperature, either in the presence or absence of IBMX (200 pM), gave identical results (data not shown). Preincubation of bovine brain membranes with compound 19 (250 nM) caused a loss of 63% of the l3H1CPX binding (Table 4). This binding was partially restored upon exposure to hydroxylamine (100-500 mM). At 37 "C, 20% of the radioligand binding relative to control level was recovered, while a t room temperature the recovery was less effective (Table 5). Varying the concentration of the isothiocyanate derivative did not improve the percent of subsequent recovery of binding. Compound 20 was prepared from the amine congener, 18,and a diisothiocyanate containing a @-hydroxypheny1)propionyl group for radioiodination (18). This diisothiocyanate, corresponding to structure 2 (Figure 1)
reversal of inhibition (% control) 37 "C rt 24 f 3 9*5 23 f 2 15, 15 ( n = 2)
'c
O
10-
c
e
2
5-
CJl
B
0'
0
50
100
150
200
2 i0
Time (min)
Figure 4. Time course for the regeneration by hydroxylamine of the specific binding of [3H]CPX to A1 receptors (percent of initial control binding) following exposure of bovine brain membranes to compound 20. The preincubation with 20 (500 nM) was carried out for 1 h at 37 "C. Following the standard washing procedure (3x 1, the membranes were treated with hydroxylamine (250 mM) for 1 h at 37 "C, and aliquots were removed at the times indicated and immediatedly diluted and washed 3 x in preparation for radioligand binding using 0.2 nM [3H]CPX. The curve is from a single regeneration experiment, in which the binding at each time point was determined three times (mean f s.e.m. shown).
in which R = CONH(CH&NHCO(CH2)2PhOH, was reported previously as a trifunctional cross-linker (11).The Ki value for 20 in the displacement of r3H1CPX in a "competitive" assay was found to be 9.3 & 2.0 nM. Preincubation of membranes with compound 20 a t a concentration of 500 nM followed by washing caused a loss of 86 f 2% ( n = 6) of the [3H]CPX binding. This binding was partially restored (19 f 3% of the fraction that was lost, n = 5) upon exposure to hydroxylamine (250 mM a t 37 "C), reaching maximal regeration after 1 h (Figure 4). Temperature of incubation and pH were varied in a n effort to improve the degree of recovery of binding following inhibition by compound 20. The hydroxylamine incubation was compared a t 37 "C for 1 h or a t 25 "C overnight. The resulting regeneration of r3H1CPX binding was comparable in both cases. An overnight incubation of membranes a t 37 "C increased the recovery (to 45%) but the binding in control membranes was diminished by 50%by 400 mM hydroxylamine. At 37 "C, a n incubation with hydroxylamine for 1-2 h gave the highest degree of recovery of r3H1CPX binding. Incubations longer than 2 h resulted in the loss of binding in control membranes. The pH of the hydroxylamine medium (37 "C for 1h) was varied from 6 to 10.5 (Table 6). Within the pH range of 7.4-9.5 the differences in recovery of [3H]CPXbinding were minor, while outside of that range less binding was recovered.
Jacobson et al.
262 Bioconjugate Chem., Vol. 6,No. 3, 1995 Table 6. Relationship between pH and the Regeneration by Hydroxylamine of r3H]CPX Binding Following Inhibition by Compound 20 reversal of inhibition (% control) PH 6 6.0 8 6.5 14 =k 3 7.4 13 f 1 8.5 15 f 3 9.5 9 f 3 10.5 a Data are means & s.e.m. of three experiments or the mean for two experiments. After incubation with the isothiocyante derivative 20 at 37 "C for 1 h, membranes were washed three times, incubated with hydroxylamine (300 mM) for 1 h at 37 "C, washed three times, and then incubated with 0.2 nM l3H1CPX(14).
DISCUSSION
A general approach for the reversible affinity labeling of receptors has been demonstrated for AI-adenosine receptors. Two sequential steps of chemical modifications of the receptor protein, i.e., affinity labeling and cleavage, result in a functionally-regenerated receptor protein (structure 4b, Figure 1) that also contains a site for a reporter group (R). Such a reporter group may consist of a radioactive or spectroscopic label, and numerous possibilities have been explored in our previous studies of trifunctional reagents (11, 20). The chain cleavage used in this study was chemically-induced, but as an alternative method photosensitive groups such as onitrobenzyl(8) may be included in protein-affinity labeling reagents. The effects on the pharmacology of a portion of the cleaved ligand being left on the receptor following restoration of the radioligand binding is the subject of ongoing studies. Thus, the regerated receptor may not be identical in binding properties to the native receptor. The cleavable portion of the ligand consists of a xanthine amine congener, in which the pharmacophore and a n amino group are separated by a cleavable, i.e., disulfide or ester, linkage (compounds 11 and 18,respectively). Cystamine, 7, was previously incorporated into thiol-cleavable cross-linking reagents for oligonucleotides (19) and in biotin avidin probes (10). In those studies the disulfide bond was easily reduced in the presence of DTT. Similarly, hydroxylamine readily cleaves ester groups, and its ability to remove a n affinity label from a fragment of the P-adrenergic receptor fragment was interpreted to indicate a n ester linkage (22). Use of these linkages in cleavable affinity labels assumes that the receptor itself is stable to the conditions needed for the cleavage reaction. In control experiments (Table 1)the native bovine AI-receptor was not denatured by moderate concentrations of DTT or high concentrations of hydroxylamine. Millimolar concentrations of DTT denatured the AI-receptor, presumably through reduction of protein disulfide bridges, as have been proposed in a receptor model (21). Nevertheless, somewhat selective reduction of the disulfide bond of the receptor-bound affinity label appears to have been accomplished in the concentration range of 0.05-0.1 M DTT. Hydroxylamine alone displaced agonist ([3H]PIA) binding from bovine AI-receptors in a noncovalent manner. Perhaps hydroxylamine in its protonated form binds to the putative Na- binding site on the second transmembrane helix of the AI-receptor, identified by a consensus sequence (21). Binding at this site would be expected to affect agonist binding adversely, but not antagonist binding, consistent with the present findings. Since the AI-receptor was more stable to hydroxylamine than to DTT, it was the ester linkage that was
selected for inclusion in a ligand, 20, containing the iodinatable (18)(p-hydroxypheny1)propionylgroup. This group has been used to incorporate a n lz5I label in a xanthine that readily cross-links to purified AI-receptors (20).
Isothiocyanate derivatives 12, 19, and 20 inhibited radioligand binding to AI-receptors in bovine brain membranes in a manner that was not reversed by repeated washing. Similar behavior was observed for the binding of m-DITC-XAC, 5, to bovine brain receptors, in which case covalent cross-linking was demonstrated by Western blot analysis (6). Hydroxylamine or DTT successfully restored binding of I3H1CPX in AI-receptors inhibited by the appropriate cleavable xanthine isothiocyanate derivative. Binding was not fully restored, but the partial reversal is sufficient to illustrate the feasibility of this approach. It may be possible to purify the cleaved and functional receptor by affinity chromatography. The AI-receptor itself was stable to a wide pH range. Within this range, hydroxylamine reversed labeling by 20 by approximately 20% of the control value. Cleavage of an ester by hydroxylamine requires the free amine. At low pH, hydroxylamine is primarily protonated, which may explain why below pH 6.5 it was not effective. The development of this approach for adenosine receptors may serve as a model for extending the method to derivatizing other G-protein coupled receptors, which have the same overall architecture, and conceivably to other biopolymers. A site-specifically labeled receptor that still binds ligand is potentially of use in the screening of drug analogs for affinity. A spectroscopic reporter group, such as a fluorescent label, present in a functional ligand binding site, may show sensitivity to ligand-bound and free states of the receptor. Thus, such a group may give a detectable signal that would report drug-receptor interactions in real time. Also, incorporation of a reactive handle, such as a thiol group, that likely results after DTT treatment of the AI receptor labeled by compound 12,offers new possibilities for derivatizing receptors. It may be possible to bind a functionalized receptor (e.g., bearing a free thiol group) to a n affinity column. Binding of the receptor to an affinity support column may also be accomplished by immobilizing group R in a derivative similar to compound 20, followed by the solubilized receptor and subsequently hydroxylamine. Such a n immobilized receptor would have many envisioned uses, such as the determination of affinity of soluble ligands by retention on a flow through column. The biospecific elution of a n adsorbed radioligand would indicate the presence of a high affinity competing ligand in solution. This scheme could potentially be used for screening libraries for active congeners (23). LITERATURE CITED (1) Newman, A. H. (1990) Irreversible ligands for drug characterization. Ann. Rep. Med. Chem. 25, 271-280. (2) Jacobson, K. A., Barone, S., Kammula, U., and Stiles, G. L. (1989) Electrophilic derivatives of purines as irreversible inhibitors of AI-adenosine receptors. J. Med. Chem. 32,10431051. (3) Barrington, W. W., Jacobson, K. A., and Stiles, G. L. (1989) Demonstration of distinct agonist and antagonist conformations of the A1 adenosine receptor. J . Biol. Chem. 264,1315713164. (4) Curtis, C. A., Wheatley, M., Bansal, S., Birdsall, N. J., Eveleigh, P., Pedder, E. K., Poyner, D., and Hulme, E. C. (1989) Propylbenzylcholine mustard labels a n acidic residue in transmembrane helix 3 of the muscarinic receptor. J . Biol. Chem. 264, 489-495. (5) Chorev, M., Feigenbaum, A., Keenan, A. K., Gilon, C., and Levitski, A. (1985) Eur. J . Biochem. 146, 9-14.
Cleavable Affinity Labeling of A,-Receptors (6) Stiles, G. A., and Jacobson, K. A. (1987). A new high affinity iodinated adenosine receptor antagonist as a radioligand photoaffinity crosslinking probe. Mol. Pharmacol. 32, 184188. ( 7 ) Jacobson, K. A., Stiles, G. L., and Ji, X.-D. Chemical modification and irreversible inhibition of striatal A2-adenosine receptors. Mol. Pharmacol. 42, 123-133. (8)Patchornik, A., Jacobson, K. A., and Strub, M. P. (1986) Photo-reversible affinity labeling. In Design a n d Synthesis of Organic Molecules Based on Molecular Recognition, Proceedings of the XVIIIth Solvay Conference on Chemistry (van Binst, G., Ed.) pp 235-241, Brussels, Springer. (9) Denny, J. B., and Blobel, G. (1984) 1251-labeledcrosslinking reagent that is hydrophilic, photoactivatable, and cleavable through a n azo linkage. Proc. Natl. Acad. Sci. U.S.A. 81, 5286-5290. (10) Haeussling, L., Ringsdorf, H., Schmitt, F. J., and Knoll, W. (1991) Biotin-functionalized self-assembled monolayers on gold: surface plasmon optical studies of specific recognition reactions. Langmuir 7, 1837-1840. (11) Boring, D. L., Ji, X.-D., Zimmet, J., Taylor, K. E., Stiles, G. L., and Jacobson, K. A., (1991) Trifunctional agents as a design strategy for tailoring ligand properties: Irreversible inhibitors of A1 adenosine receptors. Bioconjugate Chem. 2, 77-88. (12) Chong, P. C. S., and Hodges, R. S. (1981) A new heterobifunctional cross-linking reagent for the study of biological interactions between proteins. J . Biol. Chem. 256,5064-5070. (13) Garritsen, A. (1990) Molecular pharmacology of the adenosine A1 receptor. Ph.D. Thesis, Center for Bio-pharmaceutical Research, Leiden, Netherlands, 58. (14) Bruns, R. F., Fergus, J. H., Badger, E. W., Bristol, J. A., Santay, L. A,, Hartman, J. D., Hays, S. J., and Huang, C. C. (1987) Binding of the AI-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes. Naunyn Schmiedebergs Arch. Pharmacol. 335, 59-63. (15) Cheng, Y.-C., and Prusoff, W. H. (1973) Relationship between the inhibition constant (KJ and the concentration
Bioconjugafe Chem., Vol. 6,No. 3, 1995 263 of inhibitor which causes 50 percent inhibition (IC50)of an enzyme reaction. Biochem. Pharmacol. 22, 3099-3108. (16) Jacobson, K. A., Kirk, K. L., Padgett, W. L., and Daly, J. W. (1985) Functionalized congeners of 1,3-dialkylxanthines: preparation of analogues with high affinity for adenosine receptors. J. Med. Chem. 28, 1334-1340. (17) Jacobson, K. A., de la Cruz, R., Schulick, R., L. Kiriasis, Padgett, W., Pfleiderer, W., Kirk, K. L., Neumeyer, J. L., and Daly, J. W. (1988) 8-Substituted xanthines as antagonists as AI and A2-adenosine receptors. Biochem. Pharmacol. 37, 3653-3661. (18) Bolton, A. E., and Hunter, W. M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a 1251-containing acylating agent. Biochem. J . 133, 529-539. (19) Ferenz, A. E., and Verdine, G. L. (1991) Disulfide crosslinked oligonucleotides. J . Am. Chem. SOC.113, 4000-4002. (20) Jacobson, K. A., Olah, M. E., and Stiles, G. L. (1992) Trihnctional ligands: A radioiodinated high affinity acylating antagonist for the A1 adenosine receptor. Pharmacol. Commun., 1, 145-154. (21) van Galen, P. J. M., Stiles, G. L., Michaels, G., and Jacobson, K. A. (1992) Adenosine A1 and A2 receptors: Structure-function relationships. Med. Res. Rev. 5,423-471. (22) Eshdat, Y., Chapot, M.-P., and Strosberg, A. D. (1989) Chemical characterization of ligand binding site fragments from turkey P-adrenergic receptor. FEBS Lett. 246,166-170. (23) Zuckermann, R. N., Kerr, J. M., Siani, M. A., Banville, S. C., and Santi, D. V. (1992) Identification of highest-affinity ligands by affinity selection from equimolar peptide mixtures generated by robotic synthesis. Proc. Nut. Acad. Sci. U.S.A. 89, 4505-4509. (24) Jacobson, K. A., van Galen, P. J . M., Ji, X.-d., Ramkumar, V., Olah, M., and Stiles, G. L. (1993) Molecular Characterization of A1 and Aza adenosine receptors. Drug Devel. Res. 28, 226-231. BC950003E
